E-selectin (also known as ELAM-1, CD62, and CD62E) is a cytokine inducible cell surface glycoprotein cell adhesion molecule that is found exclusively on endothelial cells. E-selectin mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells, to activated endothelium (Bevilacqua, et al., “Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins,” Science 243; 1160 (1989); Graber, et al., “T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1” J. Immunol. 145: 819 (1990); Carlos, et al., “Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1” Blood 77: 2266 (1991); Hakkert, et al., “Neutrophil and monocyte adherence to and migration across monolayers of cytokine-activated endothelial cells: the contribution of CD18, ELAM-1, and VLA-4” Blood 78: 2721 (1991); and Picker, et al., “ELAM-1 is an adhesion molecule for skin-homing T cells” Nature 349: 796 (1991)).
The expression of E-selectin is induced on human endothelium in response to the cytokines IL-1 and TNF, as well as bacterial lipopolysaccharide (LPS), through transcriptional upregulation (Montgomery, et al., “Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription” Proc. Natl. Acad. Sci. 88: 6523 (1991)). E-selectin is expressed in vascular endothelial tissue where cells have been activated. Pober, J. S., et al., “Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells,” J. Immunol. 136: 1680 (1986); Bevilacqua M. P., et al., “Identification of an inducible endothelial-leukocyte adhesion molecule,” Proc. Natl. Acad. Sci. 84: 9238 (1987). Activation of vascular endothelial cells is believed, in at least some cases, to be involved in inflammatory vascular tissue damage leading to thrombosis (Fareed, J. et al., “Molecular markers of hemostatic activation. Implications in the diagnosis of thrombosis, vascular, and cardiovascular disorders,” Clin. Lab. Med. 15: 39 (1995)).
It is well-established that vascular tissue damage and thrombosis are involved in the development of strokes. Decreased supply of oxygen and nutrients from the blood to brain cells due to vascular tissue damage and thrombosis leads to the death of brain cells, the clinical manifestations of a stroke, and causes the formation of detectable spaces left by these cells, called infarctions. Strokes are a major cause of mortality in the world and account for tens of billions of dollars of medical costs in the United States alone. Although some treatments for stroke prevention are available, there is a need for more effective treatments that are applicable to a larger fraction of afflicted patients.
Structurally, E-selectin belongs to a family of adhesion molecules termed “selectins” that also includes P-selectin and L-selectin (see reviews in Lasky, “Selectins: interpreters of cell-specific carbohydrate information during inflammation” Science 258: 964 (1992) and Bevilacqua and Nelson, “Selectins” J. Clin. Invest. 91: 379 (1993)). These molecules are characterized by common structural features such as an amino-terminal lectin-like domain, an epidermal growth factor (EGF) domain, and a discrete number of complement repeat modules (approximately 60 amino acids each) similar to those found in certain complement binding proteins.
Recently, new methods and pharmaceutical formulations have been found that induce tolerance, orally or mucosally (e.g., by intranasal administration, using as tolerizers autoantigens, bystander antigens, or disease-suppressive fragments or analogs of autoantigens or bystander antigens). Such treatments are described in Wiener, H. et al., “Bystander suppression of autoimmune diseases,” WO9316724 (1993); Brigham & Womens Hospital (US), “Enhancement of the down-regulation of autoimmune diseases by oral administration of autoantigens,” WO9112816 (1991); Weiner, H. et al., “Improved treatment of autoimmune diseases by aerosol administration of auto antigens,” WO9108760 (1991); Weiner, H. et al., “Methods of treating or preventing autoimmune uveoretinitis in mammals,” WO9101333 (1991); Weiner, H. et al., “Method of treating or preventing type 1 diabetes by oral administration of insulin,” WO9206704 (1992); Hafler, D. et al., “Bystander suppression of retroviral-associated neurological disease,” WO940121 (1994); Weiner, H. et al., “Method of treating rheumatoid arthritis with type II collagen,” WO9407520 (1994); Weiner, H. et al., “Methods and compositions for suppressing allograft rejection in mammals,” WO9207581 (1992); Wucherpfenning, K. et al., “Multiple sclerosis T-cell receptor,” WO9115225 (1991); Weiner, H. et al., “Suppression of proliferative response and induction of tolerance with polymorphic class II mhc allopeptides,” WO9320842 (1993); Weiner, H. et al., “Suppression of T-cell proliferation using peptide fragments of myelin basic protein,” WO9321222 (1993); and Weiner, H. et al., “Treatment of autoimmune diseases by oral administration of autoantigens,” WO9206708 (1992).
Intravenous administration of autoantigens (and fragments thereof containing immunodominant epitopic regions) has been found to induce immune suppression through a mechanism called clonal anergy. Clonal anergy causes deactivation of only immune attack T-cells specific to a particular antigen, the result being a significant reduction in the immune response to this antigen. Thus, the autoimmune response-promoting T-cells specific to an autoantigen, once clonal anergized, no longer proliferate in response to that antigen. This reduction in proliferation also reduces the immune reactions responsible for autoimmune disease symptoms (such as neural tissue damage that is observed in MS). There is also evidence that oral administration of autoantigens (or immunodominant fragments) in a single dose and in substantially larger amounts than those that trigger “active suppression” may also induce tolerance through clonal anergy (or clonal deletion).
A method of treatment has also been disclosed that proceeds by active suppression. Active suppression functions via a different mechanism from that of clonal anergy. This method, discussed extensively in Weiner (1993), involves oral or mucosa administration of antigens specific to the tissue under autoimmune attack. These so called “bystander antigens” cause regulatory (suppressor) T-cells to be induced in the gut-associated lymphoid tissue (GALT), or bronchial associated lymphoid tissue (BALT), or most generally, mucosa associated lymphoid tissue (MALT); MALT includes both GALT and BALT. These regulatory cells are released in the blood or lymphatic tissue and then migrate to the organ or tissue afflicted by the autoimmune disease and suppress autoimmune attack of the afflicted organ or tissue.
The T-cells elicited by the bystander antigen recognize at least one antigenic determinant of the bystander antigen used to elicit them and are targeted to the locus of autoimmune attack where they mediate the local release of certain immunomodulatory factors and cytokines, such as transforming growth factor beta (TGF-β), interleukin-4 (IL-4), and/or interleukin-10 (IL-10). Of these, TGF-β is an antigen-nonspecific immunosuppressive factor in that it suppresses immune attack regardless of the antigen that triggers the attack. (However, because oral or mucosa tolerization with a bystander antigen only causes the release of TGF-β in the vicinity of autoimmune attack, no systemic immunosuppression ensues.) IL-4 and IL-10 are also antigen-nonspecific immunoregulatory cytokines. IL-4 in particular enhances Th2 response (i.e., acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells at the expense of Th1 responses). IL-4 also indirectly inhibits Th1 exacerbation. IL-10 is a direct inhibitor of Th1 responses. After orally tolerizing mammals afflicted with autoimmune disease conditions with bystander antigens, increased levels of TGF-β, IL-4, and IL-10 are observed at the locus of autoimmune attack (Chen, Y. et al., “Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis,” Science, 265: 1237-1240, (1994)). The bystander suppression mechanism has been confirmed by von Herrath et al., “Oral insulin treatment suppresses virus-induced antigen-specific destruction of beta cells and prevents autoimmune diabetes in transgenic mice,” J. Clin. Invest., 96: 1324-1331, (1996).
Although the induction of tolerance and a bystander effect has been demonstrated for a number of antigens, there remains a need to develop methods for inducing tolerance to E-selectin, and a determination of whether such induction is possible. Furthermore, there remains a need to determine whether E-selectin can be used as a bystander antigen for the induction of tolerance that provides active suppression.
This invention meets these needs by providing a method for inducing tolerance of E-selectin. Furthermore, this invention provides a method for treating stroke by the treatment of E-selectin, apparently through a bystander effect provided by E-selectin tolerance. These and other advantages, benefits, and uses of the present invention will be apparent to those of skill in the art upon a consideration of the present specification.